The modeling projects in this application focus on development and implementation of new structure based design tools for influenza HA protein specific antibodies (RP2) or vaccine antigens (RP3). These projects will use knowledge about the structure and function of human neutralizing antibodies to the HA head that we derive in Aims 1 and 2 here in RP1 in order to design new antibodies or vaccines in silico. We have access to peripheral blood cells from a diverse panels of subjects with prior natural infection, or experimental infection with vaccines encoding HA molecules with both seasonal vaccines and unusual experimental influenza subtypes, including H3variant, H5, H6, H7, H9, and H10 viruses. The immune B memory cell populations from these individuals are the ideal materials with which to search for unusual heterosubtypic antibodies. Thus, in Aim 1, we will isolate broadly heterosubtypic human mAbs to the receptor binding domain of the HA head. In Aim 2, we will determine the immunome of the responding heterosubtypic clones using high-throughput next generation sequencing of antibody gene repertoires that comprise the clonal lineages of the most heterosubtypic antibodies isolated in Aim 1. These sequences will provide an order of magnitude or more increased information on the sequences encoding heterosubtypic antibody clones. Once antibodies with unusual breadth or activity are isolated, the structure of these antibodies will be determined in complex with purified HA molecules in the Structural Core at Scripps using crystallography and single particle EM. Such structures will provide the coordinates for the modeling experiments in RP2 and RP3. RP2 will computationally design in silico maturation of antibodies to increase affinity for the HA antigen of specific virus types and use multi-state design to create antibodies that recognize HAs of multiple different clades, subtypes, groups, or even types. In RP1 Aim 3 we then will synthesize and express these novel designed antibodies and determine neutralization activity, binding affinity, and competition binding groups of designed antibodies, using a diverse HA panel and pseudotyped viruses with all type A HAs in nature. RP3 similarly will use computational methods to design structurally stable epitope-focused immunogens and experimentally test those methods by evaluating immune responses to designed model antigens. In work in RP1 Aim 3 we will validate these designs by testing the interaction of human mAbs we isolate in detailed interaction studies with the novel immunogens, to validate their likely immunogenicity. Then, we will use the novel immunogens to isolate new antibodies from subjects naturally exposed to influenza, to show that the immunogens present antigens recognized by natural immune responses